ARTICLES

PUBLISHED: 28 NOVEMBER 2016 | VOLUME: 2 | ARTICLE NUMBER: 16226

A mouse model for MERS coronavirus-induced

acute respiratory distress syndrome
Adam S. Cockrell1*, Boyd L. Yount1, Trevor Scobey1, Kara Jensen1, Madeline Douglas1, Anne Beall1,
Xian-Chun Tang2,3, Wayne A. Marasco2,3, Mark T. Heise4,5*† and Ralph S. Baric1,4*†

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel virus that emerged in 2012, causing acute
respiratory distress syndrome (ARDS), severe pneumonia-like symptoms and multi-organ failure, with a case fatality rate
of ∼36%. Limited clinical studies indicate that humans infected with MERS-CoV exhibit pathology consistent with the late
stages of ARDS, which is reminiscent of the disease observed in patients infected with severe acute respiratory syndrome
coronavirus. Models of MERS-CoV-induced severe respiratory disease have been difficult to achieve, and small-animal
models traditionally used to investigate viral pathogenesis (mouse, hamster, guinea-pig and ferret) are naturally resistant
to MERS-CoV. Therefore, we used CRISPR–Cas9 gene editing to modify the mouse genome to encode two amino acids
(positions 288 and 330) that match the human sequence in the dipeptidyl peptidase 4 receptor, making mice susceptible
to MERS-CoV infection and replication. Serial MERS-CoV passage in these engineered mice was then used to generate a
mouse-adapted virus that replicated efficiently within the lungs and evoked symptoms indicative of severe ARDS,
including decreased survival, extreme weight loss, decreased pulmonary function, pulmonary haemorrhage and
pathological signs indicative of end-stage lung disease. Importantly, therapeutic countermeasures comprising MERS-CoV
neutralizing antibody treatment or a MERS-CoV spike protein vaccine protected the engineered mice against MERS-CoV-
induced ARDS.

T
he severity of respiratory illness caused by Middle East respir-
atory syndrome coronavirus (MERS-CoV), its pandemic
potential through human-to-human respiratory transmission
and a dearth of effective treatments necessitate the development
of new MERS-CoV therapies and vaccines. Effective vaccine and
therapeutic development require preclinical animal models that
resemble the pathogenesis of human MERS-CoV infection.
Additionally, these models should: (1) include a measure of mor-
tality associated with severe respiratory disease; (2) not be con-
founded by neurological complications due to high viral loads in
the brain; (3) exhibit sustained, high-level virus replication within
the lungs of infected animals; (4) exhibit lung pathology associated
with human acute respiratory distress syndrome (ARDS); (5) main-
tain innate expression of the MERS-CoV host receptor, dipeptidyl
peptidase 4 (DPP4), to prevent perturbation of immunological
homeostasis; (6) be genetically tractable to study host genes that
regulate responses to MERS-CoV vaccines and therapeutics; and
(7) exhibit reproducibility.
Conventional non-human primate (NHP) models have been
established for MERS-CoV in both the rhesus macaque and
common marmoset1–4. NHPs are instrumental for the preclinical
development of therapeutics; however, these models are cost-
prohibitive for initial screening of large numbers of vaccine and
therapeutic candidates, challenging to work with for routine
pathogenesis studies, limited in availability and typically require
high virus challenge doses into multiple sites. Furthermore, two
recent studies have contradicted the initial studies in NHPs, which
may complicate use of the rhesus macaque5 or the common
marmoset6 model for routine vaccine or therapeutic testing.
MERS-CoV fails to replicate in traditional small-animal models
(mouse, hamster, guinea-pig and ferret) due to the inability of the
receptor-binding domain in the MERS-CoV spike protein to inter-
act with the respective DPP4 receptor7–10. In addition to acting as
the MERS-CoV receptor, DPP4 regulates T-cell activation, cytokine
function and trans-endothelial migration to sites of inflammation11.
Therefore, overexpression of DPP4 may result in immune dysregu-
lation. Effective models would therefore ideally promote functional
MERS-CoV/DPP4 interactions, with minimal perturbations of
innate DPP4 expression, signalling activity or tissue distribution.
Classical strategies to overcome receptor incompatibilities to gener-
ate susceptible mice have relied on generalized or tissue-specific
transgenic overexpression approaches to drive expression of
the human receptor (hDPP4) in the mouse12–15. Although
MERS-CoV can elicit respiratory disease in hDPP4 overexpression
models, these models exhibit a fatal central nervous system (CNS)
and systemic multi-organ disease12–14, probably due to non-specific
overexpression of the receptor throughout the animal, which com-
plicates the study of MERS-CoV-induced respiratory pathogenesis
in these models.
In this study, we used our knowledge of which determinants
allow mouse DPP4 to act as a functional MERS-CoV receptor7 by
using CRISPR–Cas9 (clustered regularly interspaced short palindro-
mic repeats and CRISPR-associated gene 9) genome editing tech-
nology to insert codons that match the human sequence at
positions 288 and 330 in the mouse Dpp4 gene. This strategy
resulted in a mouse that is permissive for MERS-CoV infection,
while maximally preserving the species-specific interaction
networks critical for DPP4 immune function. Generation of mice

1
Department of Epidemiology, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina 27599, USA. 2 Department of Cancer Immunology and
AIDS, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02215, USA. 3 Department of Medicine, Harvard Medical School,
Boston, Massachusetts 02115, USA. 4 Departments of Microbiology and Immunology, University of North Carolina-Chapel Hill, Chapel Hill, North
Carolina 27599, USA. 5 Departments of Genetics, University of North Carolina-Chapel Hill, Chapel Hill, North Carolina 27599, USA. † These authors jointly
supervised this work. * e-mail: adam_cockrell@unc.edu; mark_heisem@med.unc.edu; rbaric@email.unc.edu

NATURE MICROBIOLOGY 2, 16226 (2016) | DOI: 10.1038/nmicrobiol.2016.226 | www.nature.com/naturemicrobiology 1

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ARTICLES NATURE MICROBIOLOGY

a b c 288/330+/+ 288/330+/− C57BL/6J WT

C57BL/6J WT
Exon 10 A288 Exon 11 T330

288/330+/+

288/330+/−
Exon 10 A288 Exon 11 T330
Lung
C57BL/6J WT

Exon 10 288L Exon 11 330R

Exon 10 A288 Exon 11 T330

288/330+/− Brain

Exon 10 288L Exon 11 330R

Exon 10 288L Exon 11 330R

288/330+/+
Kidney

d 1 × 109 288/330+/+ abc

288/330+/− def
1 × 108 C57BL/6J WT
a b e
Titre (p.f.u. ml−1 per g lung tissue)

d
1 × 107 c f
e 288/330+/+ 288/330+/− C57BL/6J WT
1 × 106

1 × 105 H&E

1 × 104

1 × 103

1 × 102 LOD IHC

1 × 101

1 × 100
EMC/2012 Camel MERS icMERS MERS-0

Figure 1 | A CRISPR–Cas9 genetically engineered mouse model for MERS-CoV replication. a, C57BL/6J mice were genetically engineered using
CRISPR–Cas9 genomic editing to encode 288L and 330R in mDPP4 on one chromosome (heterozygous, 288/330+/−) or on both chromosomes
(homozygous, 288/330+/+). b, Northern blot of mDPP4 mRNA expression. c, Immunohistochemistry (IHC) of mDPP4 protein in the lungs, brain and kidneys
of individual C57BL/6J wild-type (WT), 288/330+/− and 288/330+/+ mice. d, Viral titres for MERS-CoV at 3 days post-infection from C57BL/6J WT,
288/330+/− and 288/330+/+ (all n = 4) mice infected with 5 × 105 plaque-forming units (p.f.u.) of the indicated viruses. Bar graphs show means + s.d.
Student’s t-test was used to calculate P < 0.05 for comparisons of MERS-0 with each virus in 288/330+/+ mice (a–c on graph) and 288/330+/− mice
(d–f on graph). e, Pathology of 288/330+/+, 288/330+/− and C57BL/6J WT mice at day 3 after infection with MERS-0. Lung tissue sections were stained to
examine the pathology by haematoxylin and eosin staining (H&E), or were stained by IHC to detect nucleocapsid protein from MERS-0 infection. IHC and
H&E pathology images are representative of at least three samples. Scale bars (c,e), 1 mm.

carrying a chimaeric mouse DPP4 (mDPP4) molecule patterns in the lungs, kidneys and brains of 288/330+/+ and 288/
(A288L/T330R), combined with a mouse-adapted strain of MERS- 330+/− mice reflected those observed in C57BL/6J wild-type mice
CoV, allowed us to generate a mouse model that resembles severe (Fig. 1b,c; Supplementary Fig. 2). DPP4 is central to the
MERS-CoV-induced respiratory disease without bystander neuro- maintenance of glucose homeostasis in mammals16. Blood glucose
logical disease. In parallel, we demonstrated that this model system levels were within the normal range observed in C57BL/6J wild-
can be used for the development and testing of MERS-CoV vaccines type mice, supporting the hypothesis that biological mDPP4
and therapeutics. functions were not altered in the 288/330+/+ and 288/330+/− mice
(Supplementary Fig. 2). Moreover, basal CD4+ T-cell expression
Results of interleukin-2, tumour-necrosis factor-α, interferon-γ, CD69,
A CRISPR–Cas9-generated mouse model for MERS-CoV CD25 and mDPP4 (CD26) from the 288/330+/+ and 288/330+/−
infection. We have demonstrated previously that the introduction lines was comparable to the levels observed in C57BL/6J wild-type
of two amino acids that match the human sequence at positions mice (Supplementary Fig. 3). Notwithstanding functional T-cell
288 and 330 in the mDPP4 receptor can support MERS-CoV assessment, these results suggested that minimal alteration of the
docking, entry and replication in cell culture7. These determinants 288 and 330 alleles does not alter basal T-cell activation status.
are located within exons 10 and 11 of mDPP4 on chromosome 2 Overall expression levels, expression patterns, biological function
(Fig. 1a and Supplementary Fig. 1). Therefore, we used and the immunological profiles of mDPP4 were comparable to
CRISPR–Cas9 genome editing to introduce these determinants those of C57BL/6J wild-type mice following site-specific
(A288L and T330R) into the mDPP4 receptor (Fig. 1a and modification of the 288 and 330 alleles.
Supplementary Table 1). Two lines of C57BL/6J-derived mice The 288/330+/+ and 288/330+/− mice supported efficient infection
were generated that were either homozygous (288/330+/+) or and replication of the human MERS-CoV strain HCoV-EMC/2012,
heterozygous (288/330+/−) for the chimaeric mDPP4 alleles the camel MERS strain Dromedary/Al-Hasa-KFU-HKU13/2013
(Fig. 1a). The 288/330+/+ homozygous mice encoded the 288L and a recombinant virus derived from a molecular infectious
and 330R changes on both chromosomes, thereby expressing only clone (icMERS) in the lungs, but these virus strains could not repli-
mDPP4 with both changes (Fig. 1a). The 288/330+/− heterozygous cate in C57BL/6J wild-type mice that retained the original murine
mice encoded the 288L and 330R changes on one chromosome A288 and T330 alleles (Fig. 1d). In parallel, a MERS-CoV tissue-
and the C57BL/6J wild-type amino acids, A288 and T330, on the culture-adapted variant, derived by infection of NIH/3T3 cells ecto-
other chromosome, thereby expressing both mutated and wild- pically expressing the chimaeric mDPP4 (A288L/T330R) receptor,
type mDPP4 (Fig. 1a). The innate mDPP4 expression levels and was found to encode a three amino acid insertion (RMR) and a

2 NATURE MICROBIOLOGY 2, 16226 (2016) | DOI: 10.1038/nmicrobiol.2016.226 | www.nature.com/naturemicrobiology

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NATURE MICROBIOLOGY ARTICLES
a 120 b 110

105
100
100

Weight relative to day 0 (%)

80 95
Survival (%)

90
60
85

40 80
288/330+/+, MERS-15
75
288/330+/−, MERS-15
20 288/330+/+, MERS-15
70 288/330+/+, MERS-0
288/330+/−, MERS-15 C57BL/6J WT, MERS-15
0 65
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Day post-infection Day post-infection

c 1 × 109
Day 3
1 × 108 Day 6
d i ii
1 × 107
Titre (p.f.u. ml−1 per g lung tissue)

1 × 106

1 × 105

1 × 104
iii iv
1 × 103

1 × 102 LOD
< LOD
1 × 101

1 × 100
MERS-15 MERS-0 MERS-15 MERS-15
288/330+/+ 288/330+/+ 288/330+/− C57BL/6J WT

Figure 2 | Mouse-adapted MERS-CoV causes fatal disease in 288/330+/+ mice. Mice were inoculated intranasally with 5 × 106 p.f.u. a, Mortality of
288/330+/− (n = 10) and 288/330+/+ (n = 16) mice was monitored daily up to day 6 p.i. Data show the percentage of surviving mice. b, Mouse weights
were measured daily up to day 6 p.i. for 288/330+/+ mice infected with MERS-15 (n = 16) or MERS-0 (n = 10), 288/330+/− mice infected with MERS-15 (n = 10)
and C57BL/6J wild-type (WT) mice infected with MERS-15 (n = 7). Data are daily means of the percentage weight relative to day 0 ± s.d. c, Viral lung titres for
MERS-CoV were determined at day 3 p.i. (288/330+/− + MERS-15, n = 4; 288/330+/+ + MERS-15, n = 5; 288/330+/+ + MERS-0, n = 5; C57BL/6J WT + MERS-
15, n = 4) and day 6 p.i. (288/330+/− + MERS-15, n = 4; 288/330+/+ + MERS-15, n = 4; 288/330+/+ + MERS-0, n = 5; C57BL/6J WT + MERS-15, n = 3). The limit
of detection (LOD) is indicated. Bars are means + s.d. d, Immunohistochemistry of lung sections for anti-MERS nucleocapsid at 3 days p.i. Results show
288/330+/+ mice + MERS-15 (i), 288/330+/− mice + MERS-15 (ii), 288/330+/+ mice + MERS-0 (iii) and C57BL/6J WT mice + MERS-15 (iv). IHC images are
representative of at least three samples. Scale bar (d), 1 mm.

single amino acid change (S885L) in the S2 region of the spike gene. Mouse adaptation of MERS-CoV induces severe ARDS-like disease.
This recombinantly derived virus, MERS-0, encoding the RMR and The recombinantly derived MERS-0 virus was mouse adapted by
S885L S2 mutations, demonstrated significantly enhanced replica- serial passage for 15 rounds through the lungs in 288/330+/− mice
tion both in cell culture (Supplementary Fig. 4) and in the lungs at 3-day intervals, resulting in the MERS-15 strain. Infection of
of 288/330+/+ and 288/330+/− mice (Fig. 1d; P<0.05). Despite repli- 288/330+/+ mice via the intranasal route with MERS-15 resulted
cating to significantly higher virus titres in vivo than the other iso- in ∼70% mortality (genuine mortality, rather than mice meeting
lates, MERS-0 exhibited no evidence of severe clinical disease the typical 20% weight loss cut-off associated with humane
symptoms (Supplementary Fig. 4). Lung histology demonstrated euthanasia criteria), while 100% of infected 288/330+/− mice
that nucleocapsid antigen from MERS-0 (Fig. 1e), and from the survived (Fig. 2a). However, both lines exhibited 20–25% weight
other strains (not shown), was readily detected in the lungs of loss by day 6 p.i., in contrast to MERS-0-infected 288/330+/+ mice
infected mice by immunohistochemistry, but infected lungs exhib- or MERS-15-infected C57BL/6J wild-type mice, which exhibited
ited only moderate signs of respiratory pathology and inflammation. no weight loss (Fig. 2b). Significantly higher levels of MERS-15
These results demonstrated that we had developed a MERS-CoV replication were detectable in the lungs of both 288/330+/+ and
model that could support high levels of virus replication up to day 288/330+/− mice at days 3 and 6 p.i., while MERS-0 was mostly
3 post-infection (p.i.), but that further in vivo adaptation was cleared from the lungs by day 6 p.i. (Fig. 2c,d). Similar to MER-0,
required to achieve the respiratory symptoms characteristic of MERS-15 did not replicate in C57BL/6 wild-type mice.
MERS-CoV infection in humans. Importantly, the observed decreases in survival and weight loss

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ARTICLES NATURE MICROBIOLOGY

a 50
MERS-15, 288/330+/+
MERS-15, 288/330+/−
MERS-0, 288/330+/+
** MERS-15, C57BL/6J WT
**
**
b 0.8
MERS-15, 288/330+/+
0.7 MERS-15, 288/330+/−
MERS-0, 288/330+/+
0.6 MERS-15, C57BL/6J WT
0.5

EF50 (ml s−1)

Penh(log10)

**
** ** **
5 ** 0.4
*
0.3

0.2 ** **
** * *
** ** 0.1 **
** **
0.5 0
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Day post-infection Day post-infection

c i ii d i ii iii vii

iii iv iv v vi viii

Figure 3 | Lung function in MERS-15-infected mice. a,b, Respiratory function was monitored in live mice up to day 6 p.i. using whole-body plethysmography
to measure Penh (a) and EF50 (b) in 288/330+/+ mice infected with MERS-15 (n = 9) or MERS-0 (n = 3), 288/330+/− mice infected with MERS-15 (n = 4)
and C57BL/6J wild-type (WT) mice infected with MERS-15 (n = 3). Data are daily means ± s.d. Student’s t-test was used to compare 288/330+/+ mice
infected with MERS-15 and MERS-0 (*P < 0.05; **P < 0.01). c, Pathology of lungs from infected mice at day 3 p.i. demonstrating severe inflammation for
288/330+/+ (i) and 288/330+/− (ii) mice infected with MERS-15, and moderate inflammation for 288/330+/+ mice infected with MERS-0 (iii) and C57BL/6J
WT mice infected with MERS-15 (iv). d, Pathology at day 6 p.i. infected in mice. In 288/330+/+ mice infected with MERS-15, there was severe inflammation
and oedema in the large airways (i) and alveoli (ii), and hyaline membrane formation (iii). The 288/330+/− mice infected with MERS-15 exhibited severe
inflammation throughout the parenchyma (iv), hyaline membrane formation (v) and perivascular cuffing (vi). The 288/330+/+ mice infected with MERS-0 (vii)
and C57BL/6J WT mice infected with MERS-15 (viii) exhibited mild-to-moderate inflammation. Samples were stained with haematoxylin and eosin and are
representative of at least three samples. Scale bars (c,d), 1 mm.

induced by MERS-15 were not confounded by neurological
complications from brain infection, as plaque assays for
replication-competent virus and PCR with reverse transcription
(RT-PCR) at days 3 and 6 p.i. were negative (Supplementary
Fig. 5). Moreover, quantitative RT-PCR on the same samples
demonstrated an increase of >106 detectable viral transcripts in
infected lungs compared with similarly infected C57BL/6J mice,
with no detectable viral transcripts in the brains of these mice
(Supplementary Fig. 5c).
Although mortality and weight loss provide important measures
of MERS-CoV-induced disease, these parameters do not directly
assess the impact of virus replication on respiratory function.
Therefore, to directly assess the impact of MERS-15 infection on
respiratory function in 288/330+/+ and 288/330+/− mice, we
measured respiratory function using unrestrained plethysmography,
as demonstrated previously for respiratory pathogenesis in mouse
models of severe acute respiratory syndrome (SARS) and influ-
enza17. MERS-15 elicited severe lung disease as quantified by
enhanced pause (Penh), a unitless measure that reflects airway
obstruction/restriction due to debris in the airway, and by midtidal
expiratory flow (EF50), which represents the flow rate at which 50%
of the tidal volume has been expelled in a single breath17. MERS-15
infection led to significant increases in both Penh and EF50 in
288/330+/+ and 288/330+/− mice up to day 6 p.i. compared with
288/330+/+ mice infected with MERS-0 and C57BL/6J wild-type
mice infected with MERS-15 (Fig. 3a,b), demonstrating that
MERS-15 elicited severe respiratory distress in mice carrying the
chimaeric DPP4 receptor. This was further supported by our obser-
vation of severe haemorrhage in the lungs of both 288/330+/+ and
288/330+/− mice infected with MERS-15 at days 3 and 6
(Supplementary Fig. 6), inflammatory infiltrates by day 3 (Fig. 3c)
and respiratory pathology associated with severe acute respiratory
distress, including hyaline membrane formation, intra-alveolar
oedema, perivascular cuffing and severe inflammation, at day
6 p.i. (Fig. 3d). Quantitative comparison of the lung pathology in
288/330+/+ mice infected with MERS-15 and MERS-0 demonstrated
that MERS-15 induced significant levels of pathology commensu-
rate with ARDS by day 6 p.i. (Supplementary Fig. 7). Although
we did not conduct an exhaustive assessment of all extrapulmonary
tissues in the 288/330+/+ mice, we could not detect virus replication
in the brain, even at doses of 5 × 106 plaque-forming units (p.f.u.)
up to day 6 when the humane euthanasia end points were
reached, and the pathology observed in our model was consistent
with the severe respiratory pathology associated with fatal ARDS
in the only published case study of a human MERS-CoV infection18.
Identification of MERS-CoV-adapted mutations associated with
severe respiratory disease. We anticipated that the MERS-CoV
genome would acquire mutations due to immunological pressure
and/or enhanced virus fitness during mouse adaptation. Two viral

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NATURE MICROBIOLOGY ARTICLES
a 120 b 110

105
100

Weight relative to day 0 (%)

100

80 95
Survival (%)

90
60
85

40 80
MERS-15 C1, 288/330+/+
MERS-15 C1, 288/330+/+ 75 MERS-15 C2, 288/330+/+
20 MERS-0, 288/330+/+
MERS-15 C2, 288/330+/+ 70 MERS-15 C2, C57BL/6J WT

0 65
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Day post-infection Day post-infection
c 1 × 109
Day 3
1 × 108 Day 6
Titre (p.f.u. ml−1 per g lung tissue)

1 × 107 d i ii iii

1 × 106

1 × 105

1 × 104
iv v vi
1 × 103

1 × 102 LOD
< LOD
1 × 101

1 × 100
MERS-15 C1 MERS-15 C2 MERS-0 MERS-15 C2
288/330+/+ 288/330+/+ 288/330+/+ C57BL/6J WT

Figure 4 | Clonal isolates of mouse-adapted MERS-CoV exhibit severe respiratory disease. Mice were inoculated intranasally with 5 × 106 p.f.u. a, The
mortality of 288/330+/+ mice infected with MERS-15 C1 (n = 7) or MERS-15 C2 (n = 11) was monitored daily up to day 6 p.i. Data reflect the percentage of
surviving mice. b, Mouse weights were monitored daily for 288/330+/+ mice infected with MERS-15 C1 (n = 7), MERS-15 C2 (n = 11) or MERS-0 (n = 6), and
C57BL/6J wild-type (WT) mice infected with MERS-15 C2 (n = 6). Data are daily means ± s.d. c, Viral lung titres were determined at day 3 p.i. (288/330+/+ +
MERS-15 C1, n = 4; 288/330+/+ + MERS-15 C2, n = 3; 288/330+/+ + MERS-0, n = 3; C57BL/6J WT + MERS-15 C2, n = 3) and day 6 p.i. (288/330+/+ +
MERS-15 C1, n = 4; 288/330+/+ + MERS-15 C2, n = 6; 288/330+/+ + MERS-0, n = 3; C57BL/6J WT + MERS-15 C2, n = 3). The limit of detection (LOD) is
indicated. Bars are means + s.d. d, IHC of lung sections at 3 days p.i. from 288/330+/+ mice infected with MERS-15 C1 (i) or MERS-15 C2 (ii) and stained for
nucleopcapsid. The pathology of the lungs from 288/330+/+ mice infected with MERS-15 C2 was assessed by haematoxylin and eosin staining (H&E) at day
6 p.i. and demonstrated severe inflammation (iii), oedema (iv), hyaline membrane formation (v) and perivascular cuffing (vi). All H&E images are
representative of at least three samples. Scale bar (d), 1 mm.

clones, MERS-15 clone 1 (MERS-15 C1) and MERS-15 clone 2 orf4b into orf5 (Supplementary Fig. 9). Moreover, 5′ rapid
(MERS-15 C2), were isolated by plaque purification from the amplification of cDNA ends revealed that the nucleotide at
MERS-15 heterogeneous virus population. MERS-15 C2 infection position 2 of the 5′ untranslated region was deleted in both clones
resulted in increased mortality of the mice (Fig. 4a) and (Supplementary Fig. 9). Generation of an infectious clone
significantly increased haemorrhage up to day 6 p.i. compared harbouring all of the MERS-15 C2 mutations (icMERSma1)
with MERS-15 C1 (Supplementary Fig. 8), whereas both clonal demonstrated that the disease could be reproduced with an
isolates caused 25–30% weight loss (Fig. 4b) and high levels of infectious dose of 5 × 106 p.f.u. (Supplementary Fig. 10). Decreasing
virus replication in the mice up to day 6 p.i., when humane the dose by 10-fold to 5 × 105 p.f.u. resulted in weight loss that
euthanasia end points were reached (Fig. 4c). The lung pathology paralleled that observed with 5 × 106 p.f.u.; however, the 5 × 105 p.f.u.
elicited by MERS-15 C2 resembled that obtained with the primary dose exhibited a slight decrease in mortality up to day 7 p.i.
MERS-15 virus (Fig. 4d), demonstrating similar pathologies (Supplementary Fig. 10). Although additional studies will be
including oedema, hyaline membrane formation and perivascular needed, comparative genomic analysis of the two clones, C1 and
cuffing. Sequencing of the entire MERS-15 C2 genome revealed a C2, indicated that nsP2 and Orf5 may have significant roles in
set of unique missense mutations, acquired during in vivo determining disease outcome. Therefore, the MERS-15 C2 derived
passaging, in the genes encoding the non-structural proteins, virus, combined with the 288/330+/+ mouse line, represent useful
nsP2, nsP6 and nsP8, and a large deletion in orf4b that may be tools for studying MERS-CoV pathogenesis or assessing
responsible for the enhanced disease observed with MERS-15 therapeutic countermeasures against MERS-CoV-induced ARDS.
(Supplementary Fig. 9). Sequencing of MERS-15 C1 revealed
some differences that may influence the capacity of the virus to Human monoclonal antibody 3B11 protects from MERS-CoV-
elicit the increased mortality observed with MERS-15 C2, namely elicited severe respiratory disease. Human monoclonal antibodies
mutations in nsP2 and an expanded deletion that extended from provide a robust strategy for the treatment of newly emerged viruses

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ARTICLES NATURE MICROBIOLOGY

a 120 b
105

100 100

Weight relative to day 0 (%)

95
80
Survival (%)

90
60
85

40 80

75
20 3B11 3B11
70
Isotype control Isotype control
0 65
0 1 2 3 4 5 6 0 1 2 3 4 5 6

Day post-infection Day post-infection

c 1 × 109 d
Day 3 3B11
16
1 × 108 Day 6 Isotype control
14 **
Titre (p.f.u. ml−1 per g lung tissue)

1 × 107
12
1 × 106

10
1 × 105 Penh

1 × 104 8

1 × 103 6
*

1 × 102 LOD 4
< LOD
1 × 101 2
** *
1 × 100 0
Isotype control 3B11 0 3 6
Day post-infection

Figure 5 | Human monoclonal antibody 3B11 protects mice from severe respiratory disease. The 288/330+/+ mice were intraperitoneally administered
250 µg of 3B11 human monoclonal antibody or isotype control antibody 12 h before challenge with 5 × 106 p.f.u. MERS-15 C2. a, b, Mortality (a) and mouse
weight (b) were monitored daily for mice receiving 3B11 (n = 12) or an isotype control (n = 12) up to day 6 p.i. Data show the percentage of surviving
mice (a) or daily mean weight ± s.d. (b). c, Viral lung titres were determined at day 3 p.i. (n = 6 for both 3B11- and isotype control-treated mice) and day 6 p.i.
(n = 6 for 3B11-treated mice; n = 3 for isotype control-treated mice). The limit of detection (LOD) is indicated. Bars are means + s.d. d, Lung function was
assessed by Penh at 0, 3 and 6 days p.i. for mice receiving 3B11 (n = 6 at days 0, 3 and 6) or isotype control (n = 6 at days 0 and 3; n = 3 at day 6). Data are
means + s.d. Student’s t-test was used to compare mice receiving 3B11 with those receiving the isotype control at day 3 and day 6 p.i. (*P < 0.05; **P < 0.01).

in humans. 3B11 is a human monoclonal antibody that targets the particles (VRPs) expressing MERS-CoV spike protein (spike–VRP)
receptor-binding domain of the MERS-CoV spike protein19, and is or mock vaccinated with VRPs expressing green fluorescent protein
effective in NHPs5. As the MERS-15 C2 adapted virus acquired no (GFP–VRP), boosted at 4 weeks postprime and challenged with
receptor-binding domain mutations (Supplementary Fig. 9), we MERS-15 C2 at 4 weeks postboost. All mice receiving spike–VRP
reasoned that 3B11 should protect 288/330+/+ mice from MER-15 survived and exhibited no weight loss following challenge compared
C2 challenge. Pretreating mice for 12 h with 3B11 provided 100% with GFP–VRP mock-vaccinated animals (Fig. 6a,b). Spike–VRP
protection against MERS-15 C2 challenge (Fig. 5a,b). Moreover, vaccination significantly reduced MERS-15 C2 replication in the
3B11 treatment reduced viral loads in the lungs of infected mice to lungs of infected mice as shown by both plaque titre (Fig. 6c) and
undetectable levels (Fig. 5c and Supplementary Fig. 11) and viral antigen staining (Supplementary Fig. 14), while also protecting
protected from loss of respiratory function (Fig. 5d) (Supplementary against severe respiratory disease, as assessed by measurement of
Fig. 11), pulmonary haemorrhage (Supplementary Fig. 11) and Penh (Fig. 6d) and EF50 (Supplementary Fig. 13), lung haemorrhage
severe pathological changes (Supplementary Fig. 12). In contrast, (Supplementary Fig. 13) and pathological indications of severe acute
pretreatment with isotype control antibody provided no protective respiratory disease (Supplementary Fig. 14). Neutralization of
effect. Therefore, these data convincingly demonstrated that our MERS-15 C2 with prechallenge serum from spike–VRP-vaccinated
preclinical mouse model of severe respiratory disease and mortality mice validated the presence of high-titre neutralizing antibodies in
can serve as a platform for assessing MERS-CoV therapeutics. the serum of vaccinated 288/330+/+ mice (Supplementary Fig. 13).
Our data demonstrated that the spike–VRP vaccine provoked an
Spike protein vaccines derived from Venezuelan equine adaptive immune response capable of protecting mice from a lethal
encephalitis virus replicon particles protect from lethal infection. challenge with MERS-CoV, thereby extending the utility of our
To examine vaccine efficacy in the 288/330+/+ MERS-15 C2 model, preclinical mouse model of severe respiratory disease to include
mice were vaccinated with Venezuelan equine encephalitis replicon vaccine evaluation.

6 NATURE MICROBIOLOGY 2, 16226 (2016) | DOI: 10.1038/nmicrobiol.2016.226 | www.nature.com/naturemicrobiology

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
NATURE MICROBIOLOGY ARTICLES
a 120 b
110

100 105

Weight relative to day 0 (%)

100
80
95
Survival (%)

60 90

85
40 80

75
20
Mock vaccinated (GFP−VRP) Mock vaccinated (GFP−VRP)
70
Vaccinated (spike−VRP) Vaccinated (spike−VRP)
0 65
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Day post-infection Day post-infection

c 1 × 109 d
Day 3 12 Mock vaccinated (GFP−VRP)
1 × 108 Day 6 ** Vaccinated (spike−VRP)
Titre (p.f.u. ml−1 per g lung tissue)

10
1 × 107

1 × 106
8 *
1× 105
Penh 6
1 × 104

1 × 103 4 **

1 × 102 LOD
< LOD 2
1 × 101 *

1 × 100 0
GFP−VRP Spike−VRP 0 3 6
Day post-infection

Figure 6 | Vaccination of 288/330+/+ mice with a VRP delivering MERS-CoV spike protein protects mice from challenge with MERS-CoV. The vaccination
protocol is described in Methods. a,b, After a 5 × 106 p.f.u. challenge, mortality (a) and mouse weight (b) were monitored daily for mice receiving GFP–VRP
(n = 19) or spike–VRP (n = 19) vaccine up to day 6 p.i. Data reflect the percentage survival (a) or daily mean weight ± s.d. (b). c, Viral lung titres were
determined on day 3 p.i. (n = 7 for spike–VRP and GFP–VRP) and day 6 p.i. (n = 12 for spike–VRP; n = 6 for GFP–VRP). The limit of detection (LOD) is
indicated. Bars are means + s.d. d, Lung function was assessed by Penh at 0, 3 and 6 days p.i. for mice receiving GFP–VRP (n = 12 at days 0 and 3 p.i.; n = 6
at day 6 p.i.) or spike–VRP (n = 12 at all days p.i.). Data represent means + s.d. Student’s t-test was used to compare mice receiving GFP–VRP with those
receiving spike–VRP at days 3 and 6 p.i. (*P < 0.05; **P < 0.01).

Discussion reported histopathology that included diffuse alveolar damage with

The MERS-CoV preclinical mouse model described here demon- denuding of bronchiolar epithelium, hyaline membrane formation,
strates for the first time that the CRISPR–Cas9 system can be used type II pneumocyte hyperplasia and oedema18. Furthermore,
to genetically edit a non-permissive host receptor to generate a sus- MERS-CoV antigen staining localized the virus to pneumocytes
ceptible model for an emerging infectious pathogen. The 288/330+/+ and syncytial cells18. Infection of type I and II pneumocytes can
MERS-CoV mouse model resembles the severe, and often fatal, res- lead to cell death, as observed in autopsies of patients who have
piratory distress syndrome observed in humans, and can be pre- died from severe respiratory infections with influenza virus and
vented through treatment with the 3B11 neutralizing monoclonal SARS-CoV21,22. Moreover, pneumocyte cell death has been proposed
antibody19 or a VRP-based vaccine directed against the to cause decreased respiratory function, as measured by whole-body
MERS-CoV spike protein15,20. Coupled with an inability to elicit plethysmography in a mouse model of influenza23. Commensurate
escape mutants, 3B11 has also been tested in NHPs5, making it an with these previous studies, our MERS-CoV mouse model demon-
excellent candidate for downstream human studies. However, exist- strated widespread infection of pneumocytes and pathology consist-
ing NHP models rely on quantitative RT-PCR, rather than measures ent with diffuse alveolar damage and severe respiratory disease. This
of infectious virus, to quantify viral loads, and these models do not was corroborated by decreased pulmonary function in the
reproducibly result in the severe respiratory disease or mortality MERS-CoV model, as measured by plethysmography, which may
observed in human MERS patients1–4. Therefore, the results from be associated with widespread infection and possibly the death of
our model complement the NHP 3B11 studies by directly demon- pneumocytes and airway epithelial cells. Therefore, this model
strating that 3B11 reduces levels of infectious MERS-CoV in the system provides the field with the opportunity to investigate the
lungs, while preventing severe virus-induced respiratory pathology, mechanisms that lead to MERS-CoV-induced pathology and
loss of respiratory function and mortality. severe respiratory disease, in the absence of any CNS complications.
In fatal cases of human MERS-CoV infections, individuals Other MERS-CoV mouse models have used the more traditional
exhibit severe respiratory distress requiring mechanical ventilation, method of expressing the full-length human DPP4 receptor to facili-
and the only published human autopsy from a MERS-CoV fatality tate MERS-CoV infection12–15,24. These models exhibited infection/

NATURE MICROBIOLOGY 2, 16226 (2016) | DOI: 10.1038/nmicrobiol.2016.226 | www.nature.com/naturemicrobiology 7

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
ARTICLES
replication in the lungs following intranasal administration at low
viral doses (102–105 p.f.u.), which in some cases resulted in pathol-
ogy indicative of pneumonia-like disease12–14. One limitation of the
288/330+/+ MERS-CoV model described here was the use of high
viral loads to achieve severe, and often fatal, respiratory disease.
Further adaptation may allow the use of lower infectious doses
that would produce a model of mild disease with subsequent recov-
ery at later time points, as has been described with SARS-CoV25. In
this context, it is interesting that the deletion of MERS-CoV ORF4b,
a phosphodiesterase and antagonist of RNase L activity in human
cells, appeared less critical for eliciting severe disease in rodents26,
suggesting possible species-specific modes of action in vivo.
Deletions in some SARS-CoV interferon antagonist genes and
accessory open reading frames have also yielded subtle changes in
overall virulence in vivo 27,28. However, it is important to point out
that a balance must be achieved between the virulence of mouse-
adapted virus, which enhances the model’s capacity to replicate
human disease phenotypes, and the number of mutations required
to significantly reduce the dose lethal to 50% of animals tested.
Conventional models using constitutive overexpression of the
hDPP4 MERS-CoV receptor have demonstrated widespread infec-
tion of extrapulmonary tissues including brain, kidney, liver,
spleen and heart12–14, and two of these studies indicated that the
mice exhibited multi-organ failure13,14. Furthermore, high viral
loads were detected in the brains of mice in the transgenic hDPP4
overexpression models12–14. Importantly, Li et al. 13 concluded that
“mortality correlated with brain infection, suggesting that infection
of this organ was most important for the high mortality observed in
K18-hDPP4 mice”. While these lethal models have value for vaccine
and immunotherapeutic testing29, small-molecule inhibitors that are
effective in the lung may be limited in their efficacy due to an
inability to cross the blood–brain barrier30. Similar neurological
complications have been observed in model systems for SARS that
used overexpression, or tissue-specific constitutive promoters, to
express human angiotensin 1-converting enzyme 2 (ACE2) in
mice31. While additional human pathology studies are needed to
determine the extent of extrapulmonary sites of MERS-CoV replica-
tion and their impact on MERS-CoV disease, it is clear that respir-
atory replication and pathology is an important aspect of human
MERS-CoV disease. Therefore, an important attribute of the
288/330+/+ model is that the lack of detectable virus replication in
the CNS means this model can be used to study MERS-CoV-
induced pulmonary disease without the confounding effects of
death due to CNS infection.
Recently, a debate has emerged around the safety of performing
gain-of-function (GOF) studies with highly pathogenic viruses
(such as MERS-CoV, SARS-CoV, influenza virus H5N1) (http://
www.gryphonscientific.com/gain-of-function/). As demonstrated
here, GOF studies were absolutely necessary to develop a mouse
model that reflects the ARDS pathology observed previously in
humans infected with respiratory pathogens. Importantly, the
GOF studies performed here yielded MERS-CoV strains that
reflect the complexity of clinical isolates identified recently in
humans, where deletions were identified in ORF3 and ORF4A32.
Moreover, these GOF studies have allowed us to identify mutations
in MERS-CoV proteins that may influence how MERS-CoV inter-
acts with, and possibly circumvents, host immune responses.
Nevertheless, future studies to evaluate host factors that contribute
to MERS-CoV disease will be constrained in this model, as well as
in hDPP4 expression models, by the fact that the mice must be back-
crossed to mouse lines harbouring modified endogenous genes,
such as knock-out mice. This limitation may be overcome
through additional GOF studies that facilitate MERS-CoV adap-
tation to the innate mDPP4 receptor molecule. The continued
threats from novel emerging pathogens, such as Zika virus, will
demand the rapid development of physiologically relevant animal
8 NATURE MICROBIOLOGY 2, 16226 (2016) | DOI: 10.1038/nmicrobiol.2016.226 | www.nature.com/naturemicrobiology
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
NATURE MICROBIOLOGY
models to evaluate therapeutic countermeasures, thereby necessitat-
ing virus adaptation to host immunity to achieve effective models. It
is critical that the GOF regulatory structure does not impede the
development of robust animal models of human disease, which
are essential for protecting the public health.
Methods
Viruses, cells and plaque assays. All virus stocks were prepared on Vero CCL81
cells (ATCC). CCL81 cells were maintained routinely in Dulbecco’s modified Eagle’s
medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Sigma) and
1× antibiotic/antimycotic (Gibco). All viruses were harvested in Opti-MEM medium
(Gibco) supplemented with 3% FBS, 1× antibiotic/antimycotic, 1× non-essential
amino acids (Gibco) and 1× 1 mM sodium pyruvate (Gibco). The wild-type HCoV-
EMC/2012 strain of MERS-CoV was used at passage 10 (originally provided by Bart
Haagmans (Erasmus Medical Center Rotterdam, The Netherlands) at passage 8),
icMERS was generated previously by Scobey et al. 33, MERS camel strain Dromedary/
Al-Hasa-KFU-HKU13/2013 was used at passage 5 and was provided at passage 4 by
Malik Peiris (University of Hong Kong, China) and MERS-0 was generated in the
laboratory of R.S.B. as described below. Virus titres were determined by plaque
assays on Vero CCL81 cells33. All viruses were maintained under Biosafety Level
(BSL) 3 conditions with redundant fans and personnel powered air-purifying
respirators, scrubs, Tyvek suits, Tyvek aprons and double layers of gloves.
An icMERS virus tagged with tomato red fluorescent protein33 was passaged for
ten rounds on NIH/3T3 cells (ATCC) that were generated to stably overexpress the
mDPP4 receptor containing A288L and T330R. The mDPP4 A288L/T330R
expression cassette used to generate the cell line has been described previously7.
Sequencing of the passaged virus identified an insertion of three amino acids (RMR)
after amino acid 884 in the spike protein and an S885L change. These changes were
subcloned back into the MERS-CoV infectious clone by overlap-PCR of the F
fragment with the following primers: 5′-GGTTTCCAGAAGTGTGAGCAATTA
CTGCGCG-3′, 5′-GCAGGCCTCTGCAGTCGACGGGCCCGGGATCCAA
TGCC-3′, 5′-CCTGTTTCTATATCTACTGGCAGTCG TAGAATGCGGCTTGCA
CGTAGTGCTATTGAGGATTTGC-3′ and 5′-GCAAATCCTCA ATAGCACTA
CGTGCAAGCCGCATTCTACGACTGCCAGTAGATATAGAAACAGG-3′. The F
fragment is one part of a seven-plasmid system (A, B, C, D1, D2, E and F) that
permits partitioning of the entire genome to generate infectious MERS-CoV, as
described previously33. The PCR product encoding the insertion was subcloned back
into the F fragment using the restriction enzymes MscI and BamHI and validated by
sequencing. Recombinant MERS-0 virus was used for the in vivo passage
experiments, which were GOF studies reviewed and approved by the National
Institutes of Health (NIH). Vero CCL81 and NIH/3T3 cells were originally received
from the ATCC, which indicates that cell lines are authentic and confirms that cell
lines are mycoplasma free. None of the working cell line stocks was authenticated or
tested for mycoplasma recently, although the original seed stocks used to create the
working stocks are free from contamination. Additionally, both cell lines have been
authenticated by morphological and cytopathological evaluation. Furthermore, Vero
CCL81 cells were confirmed for DPP4 overexpression by the capacity to be infected
and to replicate MERS-CoV.
Generation of mice with mDPP4 modified at positions 288 (exon 10) and
330 (exon 11). The alleles encoding amino acids 288 and 330 are shown in
Supplementary Fig. 1. Genomic engineering of these alleles with the CRISPR–Cas9
genome editing system was performed at the University of North Carolina at Chapel
Hill (UNC-CH) Animal Models Core Facility. The messenger RNA (mRNA)
encoding Cas9 endonuclease and guide RNAs (gRNAs) were based on the system
established by Mali et al.34 and prepared as described below. The gRNAs
(Supplementary Table 1) were generated by an in vitro transcription reaction using a
T7 High Yield RNA Synthesis kit (NEB), where 1 µg DraI-linearized template DNA
was used in a 20 µl reaction following the kit guidelines for short RNA transcripts.
The reaction was incubated at 37 °C overnight, followed by DNase I (RNase-free)
digestion for 15 min at 37 °C. The gRNAs were then purified using an RNeasy
column (Qiagen) following the guidelines for short RNA purification. Capped and
polyadenylated Cas9 mRNA was prepared by an in vitro transcription reaction using
an mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies). Capped mRNA
was generated with 1 µg hCas9-T7 linearized plasmid DNA in a 20 µl reaction
containing 1× NTP/ARCA Solution, 1× T7 reaction buffer and 2 µl T7 enzyme. The
reaction was incubated at 37 °C for 1 h, followed by addition of 1 µl TURBO DNase
and digestion at 37 °C for 15 min. To add a poly(A) tail to the capped mRNA, the
20 µl reaction mix from step 1 was mixed with nuclease-free water (36 µl), 5× E-PAP
buffer (20 µl), 25 mM MnCl2 (10 µl), ATP solution (10 µl) and E-PAP (4 µl) to a
final reaction volume of 100 µl. The reaction was incubated at 37 °C for 30–45 min.
The capped and polyadenylated RNA was purified by lithium chloride precipitation
and resuspended in microinjection buffer (5 mM Tris/HCl buffer, 0.1 mM EDTA,
pH 7.5).
Fertilized zygotes were collected from C57BL/6J females that had been
superovulated and mated to C57BL/6J males. Pronuclear microinjection was
performed with 50 ng µl−1 Cas9 mRNA, 50 ng µl−1 gRNA Dpp4-g59B, 25 ng µl−1
gRNA Dpp4-g88B and 50 ng µl−1 each of Dpp4-A288L-B and Dpp4-T330R-B
NATURE MICROBIOLOGY
donor oligonucleotides (Supplementary Table 1). Injected embryos were implanted
into pseudopregnant recipients, and the resulting pups were screened for alleles
encoding changes (Supplementary Fig. 1) at positions 288 and 330. Mutations at
the 288 position were detected by amplifying biopsy DNA samples with the
following primers: Dpp4-E10ScF1: 5′-GATTCTGAGCAAGCAAACACGC-3′,
and Dpp4-E10ScR1: 5′-CCACAAGGTATCCCACAGAGACG-3′. The
752 bp PCR product was sequenced with primer Dpp4-E10-SqR1:
5′-CAAGAACCACACCAATGGAAAGTC-3′. Mutations at the 330 position were
detected by amplifying biopsy DNA samples with the following primers:
Dpp4-E11ScF1: 5′-AAGTGCTGGGATTATAGGTGGTCAC-3′, and Dpp4-E11ScR1:
5′-GTGTTTACATTCTAAGTTGGGTTTCTGC-3′. The 767 bp PCR product was
sequenced with primer Dpp4-E11-SqF1: 5′- GCATGTTATCCACTGTGCCATCTC-3′.
Five of 66 live animals produced showed evidence of both the 288 and 330 modifications.
Founders with both expected modifications were backcrossed to C57BL/6J mice to
identify animals with the 288 and 330 modifications in cis. F1 animals with both the 288
and 330 modifications were then intercrossed to produce homozygous breeder pairs for
colony enrichment and downstream studies.
Mouse infections. Genetically engineered mice with a modified mDPP4 receptor
were housed and bred in accordance with guidelines established by the Department
of Laboratory Animal Medicine at UNC-CH. As the 288/330+/+ and 288/330+/− mice
are novel mouse lines developed in the laboratory of R.S.B., experiments utilized
available male and female mice that ranged from 12 to 20 weeks of age. Based on
availability at the time of each experiment, experimental and control animals were
age- and sex-matched. No blinding was used in any animal experiments, and
animals were not randomized. Sample sizes were determined from preliminary data
that would yield statistically significant differences. Mouse studies were executed
under animal BSL3 conditions as described previously35. Before viral infection, mice
were anesthetized by administering 50 µl ketamine/xylazine mixture
intraperitoneally and then infected intranasally with 50 µl virus solution containing
5 × 106 p.f.u. Incomplete infections due to bubbling of inoculum from the nasal
cavity, the inability to inhale the entire dose or inoculum going into the mouth were
noted, and these mice were considered failures and were excluded, as described
previously35. Following sedation and infection, mice were monitored daily for weight
loss and survival, as well as for signs that the animals were moribund (including
laboured breathing, lack of movement and lack of grooming). Mice that reached 20%
weight loss were placed under exception and monitored at least twice daily. Mice that
approached 30% weight loss were euthanized immediately. Mice deemed moribund
were euthanized at the discretion of the researcher. Mice were euthanized with an
isoflurane overdose followed by a secondary thoracotomy, at various time points, to
collect lung tissues. In the absence of a thoracotomy, cervical dislocation was used as
a secondary euthanasia method. All are approved methods of the Institutional
Animal Care and Use Committee (IACUC) at the UNC-CH.
Ethics statement. Mouse studies were carried out in accordance with the
recommendations for the care and use of animals by the Office of Laboratory
Animal Welfare at NIH. IACUC at UNC-CH approved the animal studies
performed here (protocol, IACUC 13-272), using a weight loss cut-off point of
∼30% for humane euthanasia. Synthetically reconstructed MERS-CoVs were
approved by the UNC-CH Institutional Biosafety Committee, which also considered
GOF research concerns before execution of these experiments.
Analysis of serum glucose levels. All blood glucose measurements were taken
following a 6 h fast. Blood glucose was measured by tail clip sampling using an
AlphaTRAK 2 glucometer (Abbott Laboratories)36. This system is designed for use
in laboratory mice, with a normal range of 111–205 mg dl−1 (6.1–11.38 mmol l−1)
blood glucose and a detection limit of 20–750 mg dl−1 (1.1–41.62 mmol l−1)
blood glucose.
Adaptation of MERS-0 in humanized mice. The recombinant virus MERS-0 was
passed through the lungs of 288/330+/− mice every 3 days for 15 passages to obtain a
MERS-CoV that was adapted to cause respiratory disease in mice (MERS-15). At
each passage, the lungs were homogenized and 50 µl lung homogenate was used for
intranasal infection of a naïve 288/330+/− mouse. The MERS-15 mouse-adapted
virus was assessed in mice by survival, weight loss, lung titre, haemorrhaging,
respiratory function and histopathology indicative of diffuse alveolar damage and
ARDS. Clonal isolates of the MERS-15 virus were obtained by plaque purification
and amplification in Vero CCL81 cells, and were sequenced to determine the
mutations acquired during mouse adaptation of the virus. All sequencing was
performed at the UNC-CH Genome Analysis Facility. MERS-15 C2 reproduced the
severe respiratory disease observed with MERS-15; therefore, all subsequent
experiments were performed with the plaque-purified MERS-15 C2.
Human monoclonal antibody 3B11 protection study. The 288/330+/+ mice were
prophylactically administered 250 µg of human monoclonal antibody 3B11 or
isotype control antibody F10 by intraperitoneal injection19 12 h before infection with
MERS-15 C2. Mice were monitored daily for weight and survival, and killed at days 3
and 6 p.i. to collect the lungs for histology, for viral titre assessment by plaque assay
and for evaluation of lung haemorrhaging. Respiratory function was measured at
NATURE MICROBIOLOGY 2, 16226 (2016) | DOI: 10.1038/nmicrobiol.2016.226 | www.nature.com/naturemicrobiology
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
ARTICLES
day 0 to establish a baseline and again at days 3 and 6 p.i. The isolation and
production of the 3B11 and F10 antibodies has been described previously19.
Spike–VRP vaccine study and virus neutralization assay. The MERS-CoV gene
encoding the spike protein and GFP gene were packaged into VRPs generated with
helper constructs from the V3526 attenuated strain of Venezuelan equine
encephalitis virus, under BSL2 conditions, as described previously20. Mice were
administered a primary vaccination of 10 µl by footpad injection of spike–VRP
(1 × 105 p.f.u.) or control GFP–VRP (1 × 105 p.f.u.). After 28 days, the mice were
boosted with the same dose of their respective VRP strain. At 28 days postboost, all
vaccinated mice were challenged with 5 × 106 p.f.u. MERS-15 C2. Mice were
monitored daily for weight and survival, and killed at days 3 and 6 p.i. to collect
lungs for histology, assessment of viral titre by plaque assay and evaluation of lung
haemorrhaging. Respiratory function was measured at day 0 to establish a baseline
and then again at days 3 and 6 p.i.
The presence of MERS-15 C2-specific serum antibodies was assessed by a plaque
reduction neutralization titre assay. Prechallenge serum samples were collected at
25 days postboost with the spike–VRP vaccine or the GFP–VRP control. Virus
neutralization assays were performed as described previously35. The percentage of
plaque reduction was calculated as: 1 − (number of plaques with spike–VRP or
GFP–VRP serum/number of plaques with serum from naïve 288/330+/+ mice) × 100.
Respiratory function. Respiratory function was measured for individual mice, at the
indicated time points, as demonstrated previously for SARS-CoV and influenza A
virus17. Briefly, individual mice were acclimated for 30 min in individual
plethysmography chambers (Buxco Systems) and each breath was then quantified
over a 5 min period. Mice were routinely randomized into different chambers to
avoid measurement biases that could result between chambers. Data for Penh and
EF50 were analysed as described previously by our group17.
Histology. Lung, brain and kidney samples were placed in 10% phosphate-buffered
formalin for >7 days at 4 °C for fixation. Fixed tissue samples were then removed
from the BSL3, placed into cassettes for embedding in paraffin and submitted to the
Lineberger Comprehensive Cancer Center Animal Histopathology Core for
processing, sectioning and staining. Tissue sections (5 µm) were stained with
haematoxylin and eosin, and MERS-CoV nucleocapsid antigen was detected using
mouse anti-MERS nucleocapsid serum at a 1:250 dilution. The nucleocapsid
antiserum was generated in the laboratory of R.S.B. using MERS-CoV nucleocapsid–
VRP particles in BALB/c mice as described previously20. Antigen was visualized
using 3,3′-diaminobenzidine staining. An Olympus DP71 camera attached to an
Olympus BX41 microscope was used to capture images with ×10 and ×40 objectives.
Histopathology was scored, blinded to infection and animal status, for airway
disease, vascular disease, parenchymal pneumonia, diffuse alveolar damage,
eosinophils and immunohistochemistry on a scale of 0–3 (0, none; 1, mild; 2,
moderate; 3, severe).
Flow cytometry analysis. Whole peripheral blood from each mouse strain was
collected in EDTA and peripheral blood mononuclear cells were purified using
density-gradient centrifugation over Ficoll-Paque PLUS (GE Healthcare Life
Sciences). Mouse lung tissues were first dissociated mechanically using scalpel blades
and then digested further using type A collagenase (Worthington Biochemical) in
medium prepared with DNase I (1 mg ml−1) for 1 h in a shaking incubator at 37 °C.
Digested lung suspensions were filtered, centrifuged and treated with ACK lysis
buffer (Gibco). Single-cell suspensions from blood and lung tissue were resuspended
at 1 × 107 cells ml−1 in RPMI 1640 medium supplemented with 10% heat-inactivated
FBS and antibiotic/antimycotic cocktail (all from Gibco), and were used
immediately for flow cytometry. Single-cell suspensions were plated in 200 µl per
well on 96-well, round-bottomed plates and stained for flow cytometry using an
Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the
manufacturer’s protocol and using antibodies specific for CD3 (clone 145-2C11; BD
Biosciences), CD4 (clone GK1.5; BD Biosciences), CD8 (clone 53-6.7; eBioscience),
CD25 (clone PC61; BD Biosciences), CD26/DPP4 (clone H194-112; Biolegend),
CD69 (clone H1.2F3; eBioscience), interferon-γ (clone XMG1.2; BD Biosciences),
tumour-necrosis factor-α (clone MP6-XT22; eBioscience) and interleukin-2 (clone
JES6-5H4; eBioscience). Cells were also treated with a fixable LIVE/DEAD
discriminator (Invitrogen). Stained cells were fixed for 20 min in 1%
paraformaldehyde, resuspended in PBS and stored at 4 °C before acquisition within
24 h. Cell populations were first gated to exclude (1) debris, (2) doublet events and
(3) dead cells before phenotypic and functional gating. All samples were stained in
parallel with controls of fluorescence – 1. A minimum of 100,000 live, singlet events
were acquired per sample, and data were analysed using FlowJo software (TreeStar).
Northern blot analysis and RT-PCR. Northern blot analysis was performed for
detection of full-length mDPP4 in the lungs of 288/330+/+, 288/330+/− and C57BL/6J
wild-type mice. Poly(A) RNA was isolated to eliminate ribosomal RNA (rRNA;
Qiagen). Equivalent amounts of RNA were resolved on 0.8% agarose gels and
transferred to nitrocellulose membrane. A biotinylated probe (5′-biotin-gATg
(biotin-dT)gCTggTgAgCTgTgCTgCTAgCgATCCCgTggTCTTCATCC-3′) was
used to detect mDPP4.
9
ARTICLES
To quantify viral and targeted host mRNAs, MERS-CoV, mDPP4 and mouse
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) RNAs were measured in
brain and lung tissue from MERS-CoV-infected mice. Briefly, tissues were removed
and placed into RNALater (Ambion) solution and stored at −80 °C until analysis by
RT-PCR. Lung tissue was homogenized in TRIzol reagent (Invitrogen) and isolated
according to the manufacturer’s instructions. Standard RT-PCR was performed with
the following primer pairs for each of the indicated RNAs: MERS-CoV subgenomic
leader sequence: 5′-CTATCTCACTTCCCCTCgTTCTC-3′ and 5′- GAATCATTG
TTAGGGTTCCG-3′; mouse GAPDH: 5′-CAACgACCCCTTCATTgACC-3′ and
5′-GCAGGGATGATGTTCTGGGC-3′; and mDPP4: 5′-TAACGACACAGGAGTG
CCGC-3′ and 5′-TCTGCTTTTTGACTACAGGG-3′. Equivalent volumes of PCR
product were resolved on gels for a non-quantitative answer (presence or absence) of
viral subgenomic transcripts in the brain and lung.
Quantitative RT-PCR was carried out on a Roche LightCycler 480 II, with
accompanying software, to analyse MERS-CoV viral RNA and mDPP4 mRNAs, and
18S rRNA as an endogenous control. All RNAs were reverse transcribed under
standard conditions in a 20 µl reaction volume using SuperScript III reverse
transcriptase (Invitrogen). MERS-CoV viral RNA was assessed using the
following primers at 900 nM in a 20 µl reaction in a SYBR Green assay with 2×
SsoAdvanced Universal SYBR Green Supermix (Biorad). The forward primer
anneals at the MERS-CoV leader sequence in the 5′ untranslated region
(5′-GAATAGCTTGGCTATCTCAC-3′) and the reverse primer anneals in the N
gene (5′-TTGTTATCGGCAAAGGAAAC-3′). PCR conditions were 45 cycles of
95 °C for 10 s, 59 °C for 10 s and 72 °C for 15 s. Standard Taqman conditions
were used to analyse expression of mDPP4 and 18S rRNAs. The following
primers were used for mDPP4: 5′-CCCCAAGACAGTGTGGATTC-3′ and
5′-GAGGATGAGCTGAGAGAGTCTATATTT-3′; probe no. 51 was used from the
Roche Universal ProbeLibrary. PCR conditions were 45 cycles of 95 °C for 15 s and
60 °C for 50 s. A 20× commercially available primer/probe set was utilized to
quantitate the 18S rRNA (Life Technologies).
Statistical analysis. All quantitative data are presented as means ± 1 s.d. Significance
between specific data sets is described in the respective figure legends and was
determined by Student’s t-test using a one-tailed or two-tailed distribution in
Microsoft Excel software.
Biosafety and biosecurity. In vivo adaptation of the MERS-0 virus in mice had been
executed before publication of the US Government’s statement on funding pause on
certain types of GOF research37. When notice to cease all in vivo passage
experiments was received, all studies to adapt the MERS-0 virus in vivo were
immediately halted. Following our formal written request for continuation, and on
receiving an exemption from the pause and approval to continue after a National
Institute of Allergy and Infectious Diseases/NIH review, the in vivo adaptation of
MERS-0 was then continued by serial passage in mice. All studies using MERS-CoV
were executed in BSL3 facilities at UNC-CH, under conditions described previously35.
The following biosafety and biosecurity paragraph is as described by
Menachery et al. 35 All work for these studies was performed with approved standard
operating procedures and safety conditions for MERS-CoV (not a select agent),
SARS-CoV (select agent) and derivatives therein. Our institutional CoV BSL3
facilities have been designed to conform to the safety requirements that are
recommended by the Biosafety in Microbiological and Biomedical Laboratories, the
US Department of Health and Human Services, the Public Health Service, the
Centers for Disease Control and the NIH. Laboratory safety plans were submitted to,
and the facility has been approved for use by, the UNC Department of
Environmental Health and Safety (EHS) and the CDC. Electronic card access is
required for entry into the facility. All workers have been trained by EHS to safely use
powered air-purifying respirators, and appropriate work habits in a BSL3 facility and
active medical surveillance plans are in place. Our BSL3 facilities contain redundant
fans, emergency power to fans, and biological safety cabinets and freezers, and our
facilities can accommodate SealSafe mouse racks. Materials classified as BSL3 agents
consist of MERS-CoV, SARS-CoV, bat CoV precursor strains and mutants derived
from these pathogens. Within the BSL3 facilities, experimentation with infectious
virus is performed in a certified Class II Biosafety Cabinet. All members of staff wear
scrubs, Tyvek suits and aprons, powered air-purifying respirators and shoe covers,
and their hands are double-gloved. BSL3 users are subject to a medical surveillance
plan monitored by the University Employee Occupational Health Clinic (UEOHC),
which includes a yearly physical, annual influenza vaccination and mandatory
reporting of any symptoms associated with coronvirus infection during periods
when working in the BSL3. All BSL3 users are trained in exposure management and
reporting protocols, are prepared to self-quarantine and have been trained for safe
delivery to a local infectious disease management department in an emergency
situation. All potential exposure events are reported and investigated by EHS and
UEOHC, with reports filed to both the CDC and the NIH.
Data availability. The data that support the findings of this study are available from
the corresponding author on request.
Received 27 April 2016; accepted 14 October 2016;
published 28 November 2016; corrected 14 July 2017
10 NATURE MICROBIOLOGY 2, 16226 (2016) | DOI: 10.1038/nmicrobiol.2016.226 | www.nature.com/naturemicrobiology
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
NATURE MICROBIOLOGY
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Acknowledgements
These studies were supported by grants from the National Institute of Allergy and
Infectious Disease of the US NIH by awards HHSN272201000019I-HHSN27200003
(R.S.B. and M.T.H.), AI106772, AI108197, AI110700 and AI109761 (R.S.B.), and U19
NATURE MICROBIOLOGY 2, 16226 (2016) | DOI: 10.1038/nmicrobiol.2016.226 | www.nature.com/naturemicrobiology
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
ARTICLES
AI100625 (R.S.B. and M.T.H.). GOF research considerations involving MERS-0 in vivo
passage in mice and the current manuscript were both reviewed and approved by the
funding agency, the NIH. The content is solely the responsibility of the authors and does
not necessarily represent the official views of the NIH. Generation of CRISPR–Cas9-
modified mice was performed at the UNC Animal Models Core Facility under the direction
of Dale Cowley.
Author contributions
A.S.C. conceived/designed, coordinated and executed the experiments, analysed the data
and wrote the manuscript. B.L.Y. developed and recovered infectious clone viruses. T.S.
completed mouse experiments. K.J. designed and completed immunological experiments.
M.D. helped establish and maintain the mouse colony and perform molecular analyses.
A.B. helped complete the mouse experiments. X.-C.T. and W.A.M. provided critical
monoclonal antibody reagents. M.T.H. and R.S.B. conceived/designed the experiments and
wrote the manuscript.
Additional information
Supplementary information is available for this paper.
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence and requests for materials should be addressed to A.S.C., M.T.H. and R.S.B.
How to cite this article: Cockrell, A. S. et al. A mouse model for MERS coronavirus-induced
acute respiratory distress syndrome. Nat. Microbiol. 2, 16226 (2016).
Competing interests
W.A.M. has a financial interest in AbViro. The other authors declare no competing
financial interests.
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